IL-1/TNF-α-activated kinase (ITAK), and methods of making and using the same

ABSTRACT

Purified and isolated IL-1/TNF-α-activated kinase (ITAK), nucleic acids encoding ITAK, processes for production of recombinant forms of ITAK, pharmaceutical compositions containing ITAK, and use of ITAK in therapies and in various assays, including assays to detect antagonists and agonists of ITAK are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. Ser. No. 09/544,794, filed 7Apr. 2000, now U.S. Pat. No. 6,541,232, which is a Divisional of U.S.Ser. No. 08/870,529, filed 06 Jun. 1997, now U.S. Pat. No. 6,080,557,which claims domestic priority to U.S. Ser. No. 60/059,979, filed 10Jun. 1996.

TECHNICAL FIELD

The invention is generally directed toward signal transduction pathwaysassociated with inflammation, and more particularly towardIL-1/TNF-α-activated kinases (ITAK)

BACKGROUND OF THE INVENTION

Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are twocytokines produced systemically and locally in response to infection,injury or immunological challenge. Based upon studies in which theaction of one (or the other) cytokine has been specifically blockaded,or in which purified cytokines have been administered, IL-1 and TNF-αhave been implicated in a number of disease processes. For example, IL-1has been implicated in inflammatory diseases including rheumatoidarthritis and other degenerative joint diseases, inflammatory boweldisease, type I diabetes, psoriasis, Alzheimer's disease, and allergy.Overproduction of TNF-α has likewise been implicated in diseases such asreperfusion injury, rheumatoid arthritis, cardiovascular disease,infectious disease such as HIV infection and HIV-induced neuropathy,allergic/atopic diseases, inflammatory disease/autoimmunity, malignancy,transplant difficulties including organ transplant rejection orgraft-versus-host disease, cachexia, and congenital, dermatologic,neurologic, renal, toxicity and metabolic/idiopathic diseases. Aparticular case where the two cytokines are thought to actsynergistically is in the induction of the Systemic InflammatoryResponse Syndrome.

Because the consequences of uncontrolled production of IL-1 and TNF-αcan be severe, considerable effort has been expended on therapies thatwould limit the production or activity of one, or preferably both, ofthe cytokines. The prevailing therapy has been to administer proteinsthat bind specifically to the circulating cytokines, thus preventingthem from interacting with their cellular receptors. Typically theseprotein-based therapeutics are antibodies or ‘soluble’ receptors (i.e.,recombinant versions of the natural cellular receptors which lacktransmembrane and signaling domains). An additional protein-basedtherapeutic is the IL-1 receptor antagonist protein (IL-1ra), whichcompetes for binding to the same cellular receptors as the agonist formsof IL-1, but does not elicit a cellular signal.

The effectiveness of all three types of protein-based therapy is limitedbecause occupation of even a very small number of IL-1 or TNF-αreceptors by IL-1 or TNF-α generates a cellular response (and thereforethe harmful effects described above). It is therefore necessary tomaintain relatively high levels of anti-cytokine antibody, solublereceptor or antagonist protein in order to drive the equilibrium infavor of complex formation (i.e., to effectively prevent binding of IL-1or TNF-α to their respective receptors). Another drawback to suchprotein-based therapeutics is that each therapeutic is selective foronly one of the two cytokines. Thus, large doses of a multitude oftherapeutics must be administered to a patient in order to attempt tocontrol IL-1 and TNF-α production.

Although the biological effects of TNF-α and IL-1 are quite similar, thestructures of the cytokines, and the structure of their receptors, arevery different. IL-1 and TNF-α appear to have overlapping biologicalactivities because the binding of each cytokine to its receptor appearsto affect similar post-receptor signal transduction pathways. Manydetails of these pathways are unclear.

For example, although both cytokines activate the transcription factorsNF-κB and AP-1, which leads to the regulated transcription of a widevariety of genes, the particular receptor-proximal effector moleculesthat regulate this process is unclear. Additionally, both cytokines havebeen reported to cause the activation of sphingomyelinases andphospholipases that generate, respectively, ceramide and arachidonicacid. Both cytokines also activate members of the mitogen-activatedprotein kinase (MAPK) family including ERK1, ERK2, and thestress-activated kinases JNK-1 and p38. This family of kinases isactivated, to varying extent, by a wide range of hormones, growthfactors, heavy metals, protein synthetic inhibitors and ultravioletlight and therefore activation of such kinases cannot be consideredunique to the IL-1/TNF-α signal transduction pathway.

In addition to items activated by both IL-1 and TNF-α, IL-1 has beenreported to specifically activate the IL-1 receptor associated kinase,IRAK, (Cao, Henzel and Gao, Science 271:1128 (1996)). The cytoplasmicdomains of TNF receptors have also been reported to interact with othersignal transduction molecules such as TRAF1 and TRAF2, FADD, MORT andTRADD. Such TNF-α receptor-interacting proteins also appear capable ofinteracting with an extended receptor family, including those thatmediate quite distinct cellular responses such as the T- and B-cellactivator CD40 and a mediator of apoptosis, fas. (Tewari and Dixit,Curr. Opin. Genet. Dev. 6:39, 1996; Lee et al., J. Exp. Med. 183:669,1996).

While certain cellular responses may be elicited by IL-1, TNF-α, orother mediators, the only known signaling event that appears to beuniquely induced by IL-1 or TNF-α, but no other defined stimulus, is aprotein serine/threonine kinase activity that could be detected in vitroby its ability to phosphorylate β-casein. Guesdon et al., J. Biol. Chem.268:4236 (1993); Biochem. J. 304:761 (1994). This β-casein kinaseactivity was induced in fibroblasts and other connective-tissue derivedcells by IL-1 and TNF-α but not by 21 other agents tested. The structureof the β-casein kinase was not elucidated in this report.

However, there has gone unmet a need for substances and/or methods thatprovide either repression or stimulation of intracellular effects ofboth IL-1 and TNF-α. There has also gone unmet a need for substances andmethods that provide interaction(s) with the post-receptor pathway(s) ofIL-1 and TNF-α, as well as substances and methods that provideopportunities to detect agonists and/or antagonists to IL-1 or TNF-α,including single compounds that act as an agonist or antagonist to bothIL-1 and TNF-α. The present invention provides these and other relatedadvantages.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid and amino acid sequences ofprotein kinases, preferably human, that interact with at least onepost-receptor intracellular pathway of both IL-1 and TNF-α. Such kinasesare referred to herein as IL-1/TNF-α-activated kinase (ITAK). Suchkinases are induced as enzymatically active kinases capable ofphosphorylating specific substrate proteins by treatment of suitablecells with IL-1 or TNF-α. The present invention further providescompositions and methods for the isolation and purification of nucleicacid molecules encoding ITAK. Also disclosed herein are methods forexpressing and purifying ITAK, as well as specific assays for thedetection of inhibitors or stimulators of ITAK activity, which wouldhave utility as antagonists or agonists of IL-1 and TNF-α.

In addition, the present invention is directed to isolated nucleic acidsencoding ITAK and to vectors, including expression vectors, capable ofexpressing ITAK, preferably from a cDNA encoding ITAK. The presentinvention includes host cells containing such expression vectors, andprocesses for producing ITAK by culturing such host cells underconditions conducive to expression of ITAK, and preferably thepurification of ITAK, including in industrial quantities. In part due tosuch purification of ITAK, the invention is also directed to antibodies,preferably monoclonal antibodies, specific for ITAK.

The present invention is also directed to assays utilizing ITAK toscreen for potential inhibitors or stimulators of ITAK activity, forexample as a means of blocking a signal transduced in response to IL-1or TNF-α. Further, methods of using ITAK in the design of inhibitors ofITAK activity are also disclosed.

In particular, in one aspect, an isolated nucleic acid molecule encodingan IL-1/TNF-α-activated kinase (ITAK) such as a human ITAK, or variantthereof, is provided. In one embodiment, the isolated nucleic acidmolecule comprises the sequence of nucleotides in SEQ ID:NO 1, fromnucleotide number 1 to nucleotide number 2940. This isolated nucleicacid molecule encodes a protein having the amino acid sequence of SEQID:NO 2. In a related embodiment, nucleic acid molecules encoding ITAKvariants are provided, including the Lys81→Ala substituted ITAK variant.Within a related aspect, an isolated ITAK or variant thereof isprovided.

Within other related aspects, recombinant vectors, including recombinantexpression vectors comprising a promoter operably linked to ITAK-codingsequences are provided. The invention further provides host cellscontaining any such recombinant vectors.

In still another aspect, the invention provides a nucleic acid probe ofat least 15 nucleotides in length which is capable of specificallyhybridizing to a nucleic acid sequence encoding an IL-1/TNF-α-activatedkinase (ITAK).

Within yet another aspect of the invention, a method of screening for anagent that modulates the kinase activity of an IL-1/TNF-α-activatedkinase (ITAK) is provided, comprising: (a) contacting a candidate agentwith biologically active ITAK under conditions and for a time sufficientto allow the candidate agent to modulate the kinase activity of theITAK; and (b) measuring the ability of the candidate agent to modulatethe ITAK kinase activity. Within one embodiment, the method furthercomprises isolating the candidate agent.

Within another aspect of the invention, a method for determining whethera selected agent is an IL-1/TNF-α-activated kinase (ITAK) agonist isprovided, comprising: (a) exposing the selected agent to an unstimulatedITAK response pathway under conditions and for a time sufficient toallow a stimulation of the pathway; and (b) detecting stimulation of theresponse pathway and therefrom determining the presence of an ITAKagonist. Within a related aspect, a method for determining whether aselected agent is an IL-1/TNF-α-activated kinase (ITAK) agonist isprovided, comprising: (a) measuring the ITAK kinase activity of an ITAKresponse pathway; (b) exposing the selected agent to the measured ITAKresponse pathway; and (c) detecting increased ITAK kinase activity inthe response pathway.

Within still another aspect of the invention, a method for determiningwhether a selected agent is an IL-1/TNF-α-activated kinase (ITAK)antagonist is provided, comprising: (a) exposing the selected agent toan ITAK response pathway in the presence of an ITAK agonist underconditions and for a time sufficient to allow a decrease in stimulationof the pathway; and (b) detecting a decrease in the stimulation of theresponse pathway relative to the stimulation of the response pathway bythe ITAK agonist alone, and therefrom determining the presence of anITAK antagonist. Utilizing such methods, ITAK agonists and ITAKantagonists are provided.

Within yet another aspect, an ITAK phosphorylation substrate peptideacceptor sequence that is not mammalian β-casein and that can bephosphorylated by isolated ITAK at a rate of at least 40 nmol,preferably at least 80 nmol, even more preferably at least 98 nmolphosphate/mg protein/minute is provided.

Within still other aspects of the invention, a method for detectingIL-1/TNF-α-activated kinase (ITAK) activity is provided, comprising: (a)contacting ITAK with an ITAK phosphorylation substrate peptide acceptorsequence that is not mammalian β-casein in the presence of ATP underconditions and for a time sufficient to allow transfer of a γ-phosphategroup from an ATP donor to the ITAK phosphorylation substrate peptideacceptor sequence; and (b) measuring the incorporation of phosphate bythe ITAK phosphorylation substrate peptide acceptor sequence. Within oneembodiment, the ATP is γ-(³²P)-ATP. In related embodiments of theinvention, the ITAK phosphorylation substrate peptide acceptor sequencehas the amino acid sequence:Arg-Arg-Arg-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met-His-Gln-Pro-His-Gln.(SEQ ID:NO 3)

Within another aspect of the invention, a method for treating an IL-1-or TNF-α-mediated inflammatory disorder is provided, comprisingadministering to a patient a therapeutically effective amount of an ITAKantagonist. The invention further provides kits for detecting ITAK in asample, comprising: an ITAK phosphorylation substrate peptide acceptorsequence that is not mammalian β-casein and that can be phosphorylatedby isolated ITAK at a rate of at least 40 nmol, preferably at least 80nmol, even more preferably at least 98 nmol phosphate/mg protein/minute;and a means for measuring phosphate incorporated by the ITAKphosphorylation substrate peptide acceptor sequence.

The invention further provides methods for identifying gene productsthat associate with ITAK, comprising: (a) introducing nucleic acidsequences encoding an ITAK polypeptide into a first expression vectorsuch that ITAK sequences are expressed as part of a fusion proteincomprising a functionally incomplete first portion of a protein that isessential to the viability of a host cell; (b) introducing nucleic acidsequences encoding a plurality of candidate gene products that associatewith ITAK into a second expression vector such that any candidate geneproducts are expressed as part of a fusion protein comprising a secondfunctionally incomplete portion of the protein that is essential to theviability of the host cell; (c) introducing the first and secondexpression vectors into a host cell under conditions and for a timesufficient such that host cell survival is dependent upon reconstitutionof both the first and second functionally incomplete portions of theprotein into a functionally complete protein; and (d) identifyingsurviving host cells, and therefrom determining the nucleic acidsequences encoding candidate gene products that associate with ITAK inthe second expression vector.

In one embodiment of this aspect of the invention, the host cell is ayeast host cell. In another embodiment of this aspect of the inventionthe yeast is yeast strain Y190. In related embodiments, the protein thatis essential to the viability of the host cell is the modular yeasttranscription factor GAL4. In another related embodiment thefunctionally incomplete first portion of a protein that is essential tothe viability of the host cell comprises the N-terminal 147 amino acidsof the modular yeast transcription factor GAL4, while in anotherembodiment the functionally incomplete second portion comprises theC-terminal 114 amino acids of the modular yeast transcription factorGAL4. Within yet another embodiment, the functionally incomplete firstportion of a protein that is essential to the viability of the host cellcomprises the DNA binding domain of a modular transcription factor. In arelated embodiment, the functionally incomplete second portion of aprotein that is essential to the viability of a unicellular hostcomprises a transcriptional activation domain of a modular transcriptionfactor.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. Various references are set forth herein that describe certainprocedures or compositions (e.g., plasmids, etc.). All references citedherein are incorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict a representative human ITAK nucleotide sequence andcorresponding amino acid sequence (SEQ ID:NO 8).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides compositions and methodsfor the isolation and purification of ITAK proteins, which proteins havekinase activities that are specifically induced by exposure ofappropriate cells to IL-1 or TNF-α. Inhibitors of IL-1 signaltransduction have clinical utility in treating various inflammatory andimmune disorders characterized by over-production or unregulatedproduction of IL-1, such as allergy, rheumatoid arthritis, inflammatorybowel disease, psoriasis, and Alzheimer's disease. Inhibition of TNF-αsignaling also has clinical utility in treating conditions characterizedby over-production or unregulated production of TNF-α, such as SystemicInflammatory Response Syndrome, reperfusion injury, cardiovasculardisease, infectious disease such as HIV infection and HIV neuropathy,inflammatory disease/autoimmunity, allergic/atopic diseases, malignancy,transplants including organ transplant rejection or graft-versus-hostdisease, cachexia, congenital, dermatologic, neurologic, renal,toxicity, and metabolic/idiopathic diseases. The disclosure herein of acDNA that encodes ITAK provides methods and compositions suitable forthe inhibition of IL-1 and/or TNF-α, as well as a variety of otheradvantages.

Applicants' discovery of ITAK enables, among other things, constructionof vectors, including expression vectors, comprising nucleic acidsequences encoding ITAK, host cells containing such vectors (for examplevia transfection or transformation), the production of ITAK, includingindustrial amounts of ITAK, and antibodies immunoreactive with ITAK. Inaddition, understanding of the mechanism by which ITAK functions in IL-1and TNF-α signaling enables the design of assays to detect inhibitors ofIL-1 and/or TNF-α activity.

As used herein, the term “ITAK” refers to a genus of polypeptides havingkinase activities that are specifically induced by exposure of ITAKsource cells to IL-1 or TNF-α, and that are capable of thephosphorylation of dephosphorylated bovine β-casein, or thephosphorylation of other suitable peptide or polypeptide substratesidentified by their structural homology to phosphorylation acceptorsites of bovine β-casein. In general, such activities are not induced byPMA, 10% fetal calf serum, PDGF, bradykinin, EGF, TGF-β1, bFGF,interferon-β, interferon-β, histamine, prostaglandin E₂, forskolin,A23187, 44° C. heat shock or sodium arsenite (Guesdon et al., Biochem.J. 304:761 (1994)). Unless otherwise stated, ITAK also refers tovariants and derivatives thereof. In a preferred embodiment, ITAKincludes proteins having the amino acid sequence 1-979 of SEQ ID:NO 2,as well as proteins having a high degree of sequence homology (typicallyat least 90% sequence identity, preferably at least 95% identity, andmore preferably at least 98% identity) with such amino acid sequences.ITAK also includes the gene products of the nucleotides 1-2940 of SEQID:NO 1 and the amino acid sequences encoded by these nucleotides, aswell as the gene products of other ITAK-encoding nucleic acid molecules.Such proteins and/or gene products are preferably biologically active.Full length ITAK has a molecular weight of approximately 110-125 kD asdetermined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). ITAKalso includes the nucleic acid molecules encoding ITAK.

An “ITAK variant” as used herein refers to a polypeptide substantiallyhomologous to native ITAK, but which has an amino acid sequencedifferent from that of native ITAK (human, rabbit, murine or othermammalian species) because of one or more naturally or non-naturallyoccurring deletions, insertions or substitutions. The variant amino acidsequence preferably is at least about 80% identical to a native ITAKamino acid sequence, more preferably at least about 90% identical, andfurther preferably at least about 95% identical. The percent identitymay be determined, for example, by comparing sequence information usingthe GAP computer program, version 6.0 described by Devereux et al.(Nucl. Acids Res. 12:387, 1984) and available from the University ofWisconsin Genetics Computer Group (UWGCG). The GAP program utilizes thealignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970),as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Thepreferred default parameters for the GAP program include: (1) acomparison matrix (containing a value of 1 for identities and 0 fornon-identities) for nucleotides, and the weighted comparison matrix ofGribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353-358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

One class of ITAK variants is based on the tendency of protein kinasesto contain a lysine residue in catalytic subdomain II that isadvantageous for maximal enzymatic activity. In particular, mutation ofthis lysine residue (corresponding to position 81 in the ITAK disclosedin SEQ ID:NO 2) leads to loss of catalytic function in protein kinases.Thus, such a mutant (preferably recombinant) kinase can exert a“dominant negative” phenotype when overexpressed in cells, therebypreventing signalling through the biochemical pathway in which thewild-type ITAK normally functions. Such a variant, for example ITAK A81in which alanine is substituted for lysine-81, can be particularlyadvantageous for therapeutic uses in the inhibition of the IL-1 or TNF-αsignalling, as discussed further below. Other ITAK variants that lackthe protein kinase activity of ITAK, such as ITAK variants having aminoacid substitutions other than the Lys→Ala substitution of ITAK A81 andincluding amino acid deletions, insertions, or substitutions, areencompassed within ITAK variants of the invention.

ITAK variants can comprise conservatively substituted sequences, meaningthat a given amino acid residue is replaced by a residue having similarphysicochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg, Glu and Asp, or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown. Naturally occurring ITAK variants are also encompassed by theinvention. Examples of such variants are proteins that have amino acidsubstitutions, result from alternate mRNA splicing events, or resultfrom proteolytic cleavage of the ITAK protein, wherein the proteolyticfragments retain the biological activity of ITAK. Variationsattributable to proteolysis include, for example, differences in the N-or C-termini of naturally-occurring ITAK as isolated from cells ortissues, or similar variations detectable upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the ITAK protein (generally from 1-5 terminal aminoacids).

A “nucleotide sequence” refers to a polynucleotide molecule in the formof a separate fragment or as a component of a larger nucleic acidconstruct, that has been derived from DNA or RNA isolated at least oncein substantially pure form (i.e., free of contaminating endogenousmaterials) and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,1982, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).Such sequences are preferably provided in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA may be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The term “isolated” as used herein means an ITAK protein that has beenseparated from a source cell, whether recombinant or non-recombinant,such that the ITAK protein comprises at least about 90% of the proteincontent of the composition based on the staining pattern of thecomposition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using the stain Coomassie blue. Typically, the purifiedprotein comprises at least about 92% of the protein content, preferablyat least about 94% of the protein content, further preferably at leastabout 96% of the protein content, and even more preferably at leastabout 98% of the protein content. In alternative embodiments, no other(undesired) protein is detected pursuant to SDS-PAGE analysis followedby Coomassie blue staining, and preferably no other (undesired) proteinis detected pursuant to SDS-PAGE analysis followed by silver staining.An “isolated” nucleic acid molecule means a nucleic acid molecule thatencodes ITAK, as discussed further herein, and that has been isolatedfrom its source cell. Additionally, an ITAK gene (or fragment thereof,or variant, etc., also as discussed herein) is considered isolated if ithas been separated from its biological source cell nucleic acidmolecule, such as a chromosome. Such an isolated gene can be containedwithin a recombinant nucleic acid molecule.

The term “biologically active” as used herein to refer to ITAK, meansITAK that is capable of phosphorylating a dephosphorylated bovineβ-casein, or of phosphorylating synthetic peptide substrates (such asITAK phosphorylation substrate peptide acceptor sequences provided bythe invention) having substantial similarity to one or more of thephosphorylation acceptor sites of bovine β-casein. One such substrate isthe polypeptide RRRHLPPLLLQSWMHQPHQ (SEQ ID:NO 3). Preferred conditionsfor phosphorylating a dephosphorylated bovine β-casein can be found inGuesdon et al., J. Biol. Chem. 268:4236 (1993); Guesdon et al., Biochem.J. 304:761 (1994), while preferred conditions for the phosphorylation ofa synthetic polypeptide RRRHLPPLLLQSWMHQPHQ (SEQ ID:NO 3) can be foundin Example 1. In vitro phosphorylation of substrate peptides by ITAK maybe adapted to high-throughput screens, for example, by scintillationproximity assays (SPA). Thus, means for measuring phosphate incorporatedvia ITAK into β-casein or into ITAK phosphorylation substrate peptideacceptor sequences include detection of incorporated ³²P; SPA detectionmethods known in the art; fluorometric, colorimetric, orspectrophotometric measurements; immunochemical detection, for exampleby use of antibodies specifically reactive with phosphorylated aminoacids or peptides; or related detection methods known to those skilledin the art.

An isolated ITAK according to the invention may be produced byrecombinant expression systems as described below or may be producedfrom naturally occurring cells. ITAK can also be substantially purified,as indicated by a series of phosphoproteins that migrate as 110 kD to125 kD components in SDS-polyacrylamide gel electrophoresis (SDS-PAGE),and that have identical amino acid sequences as indicated by peptidemaps and partial sequence analysis. One process for producing ITAKcomprises culturing a host cell transformed with an expression vectorcomprising a DNA sequence that encodes ITAK under conditions sufficientto promote expression of ITAK. ITAK is then recovered from culturemedium or cell extracts, depending upon the expression system employed.As is known to the skilled artisan, procedures for purifying arecombinant protein will vary according to such factors as the type ofhost cells employed and whether or not the recombinant protein issecreted into the culture medium. For example, when expression systemsthat secrete the recombinant protein are employed, the culture mediumfirst may be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. Following the concentration step, the concentratecan be applied to a purification matrix such as a gel filtration medium.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred. Finally, one or morereversed-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel having pendantmethyl or other aliphatic groups) can be employed to further purifyITAK. Some or all of the foregoing purification steps, in variouscombinations, are well known and can be employed to provide an isolatedand purified recombinant protein.

In addition to recombinantly producing ITAK, ITAK may be isolated andpurified from any one of the following cell lines: C122 (Sims et al.,Proc. Nat. Acad. Sci. USA 86:8946-8950, 1989), HUT102 (ATCC TIB162), KB(ATCC CCL17), Raji (ATCC CCL86), SK-Hep-1 (ATCC HTB52 and WI-26 (ATCCCCL95.1). Other sources for ITAK may be used, and ITAK may also be foundin other types of cells that produce, or respond to IL-1 or TNF-α.Production of ITAK by a candidate cell can be detected, for example,using the assays discussed herein, such as the assays described above inrelation to the determination of ITAK biological activity and in Example1, and/or via appropriate nucleic acid hybridization assays. Once asource cell, or cell line, for ITAK is identified, ITAK may be isolatedand purified by first optionally stimulating the source cells with IL-1or TNF-α. When desired, such stimulation can be done using techniquesthat are well-known in the art. IL-1 is used preferably at 1-50 ng/mland TNF-α is used preferably at 20-200 ng/ml. (Guesdon et al. 1993,1994) The cells are then harvested, washed and cytoplasmic proteinsextracted according to conventional procedures.

Partially purified ITAK occurs as a high molecular weight complexof >350 kD that may contain specifically associating species importantto the regulation of ITAK activity. ITAK may also be modified to createITAK derivatives by forming covalent or aggregative conjugates withother chemical moieties, such as glycosyl groups, polyethylene glycol(PEG) groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of ITAK may be prepared by linking the chemical moieties tofunctional groups on ITAK amino acid side chains or at the N-terminus orC-terminus of an ITAK polypeptide. Other derivatives of ITAK within thescope of this invention include covalent or aggregative conjugates ofITAK or its fragments with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.For example, the conjugate may comprise a signal or leader polypeptidesequence (e.g., the α-factor leader of Saccharomyces) at the N-terminusof an ITAK polypeptide. The signal or leader peptide co-translationallyor post-translationally directs transfer of the conjugate from its siteof synthesis to a site inside or outside of the cell membrane or cellwall.

It is possible to utilize an affinity column comprising an ITAK-bindingprotein, for example an ITAK-binding antibody, to affinity-purifyexpressed ITAK polypeptides. ITAK polypeptides can be removed from anaffinity column using conventional techniques, e.g., in a high saltelution buffer and then dialyzed into a lower salt buffer for use or bychanging pH or other components depending on the affinity matrixutilized.

Variants and derivatives of native ITAK that retain the desiredbiological activity may be obtained by mutations of nucleotide sequencescoding for native ITAK polypeptides. Alterations of the native aminoacid sequence may be accomplished by any of a number of conventionalmethods. Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene whereinpredetermined codons can be altered by substitution, deletion orinsertion. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity arealso encompassed by the invention. For example, sequences encoding Cysresidues that are not essential for biological activity can be alteredto cause the Cys residues to be deleted or replaced with other aminoacids, preventing formation of incorrect intramolecular disulfidebridges upon renaturation. Other equivalents can be prepared bymodification of adjacent dibasic amino acid residues to enhanceexpression in yeast systems in which KEX2 protease activity is present.EP 212,914 discloses the use of site-specific mutagenesis to inactivateKEX2 protease processing sites in a protein. KEX2 protease processingsites are inactivated by deleting, adding or substituting residues toalter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence ofthese adjacent basic residues. Lys-Lys pairings are considerably lesssusceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg toLys-Lys represents a conservative and preferred approach to inactivatingKEX2 sites.

Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native ITAKnucleotide sequences disclosed herein under conditions of moderate orhigh stringency, and which encode biologically active ITAK, and theircomplements. As used herein, conditions of moderate stringency, as knownto those having ordinary skill in the art, and as defined by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Ed. Vol. 1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989), include use of aprewashing solution for the nitrocellulose filters 5× SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0), hybridization conditions of 50% formamide, 6× SSC at42° C. (or other similar hybridization solution, or Stark's solution, in50% formamide at 42° C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. Conditions of high stringency are defined ashybridization conditions as above, and with washing at 68° C., 0.2× SSC,0.1% SDS. The skilled artisan will recognize that the temperature, saltconcentration, and chaotrope composition of hybridization and washsolutions may be adjusted as necessary according to factors such as thelength and nucleotide base composition of the probe.

Due to the known degeneracy of the genetic code wherein more than onecodon can encode the same amino acid, a DNA sequence may vary, forinstance from that shown in FIG. 1 and still encode an ITAK protein,such as one having the amino acid sequence of SEQ ID:NO 2. Such variantDNA sequences may result from silent mutations (e.g., occurring duringPCR amplification), or may be the product of deliberate mutagenesis of anative sequence.

The invention thus includes equivalent isolated nucleic acid sequencesencoding biologically active ITAK, including those selected from: (a)nucleic acid molecules derived from the coding region of a nativemammalian ITAK gene; (b) nucleic acid molecules selected from the groupconsisting of nucleotide sequences SEQ ID:NO 1 and SEQ ID:NO 8; (c)nucleic acid molecules capable of hybridization to a nucleic acidmolecule of (a) (or their complementary strands) under conditions ofmoderate stringency and which encode ITAK; and (d) nucleic acidmolecules which are degenerate, as a result of the genetic code, withrespect to a nucleic acid molecule defined in (a), (b) or (c) and whichcodes for ITAK. Preferably, the nucleic acid molecule is DNA, andfurther preferably the ITAK is biologically active. ITAK proteins andgene products encoded by such equivalent nucleic acid sequences areencompassed by the invention.

Nucleic acid molecules that are equivalents to the DNA sequence of FIG.1, SEQ ID:NO 1 will hybridize under moderately stringent conditions tothe double-stranded native DNA sequences that encode polypeptidescomprising amino acid sequences of SEQ ID:NO 2. Examples of ITAKsencoded by such DNA, include, but are not limited to, ITAK fragments andITAK proteins comprising inactivated KEX2 protease processing site(s),or conservative amino acid substitution(s), including those describedabove. ITAK proteins encoded by DNA derived from other mammalianspecies, wherein the DNA will specifically hybridize to the complementof the cDNA of FIG. 1 or SEQ ID:NO 1 or SEQ ID:NO 8 are alsoencompassed.

ITAK polypeptide conjugates can comprise peptides added to ITAK tofacilitate purification and identification of ITAK. Such peptidesinclude, for example, poly-His or the antigenic identification peptidesdescribed in U.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology6:1204, 1988. ITAK fusion proteins may further comprise immunoglobulinconstant region polypeptides added to ITAK to facilitate purification,identification, and localization of ITAK. The constant regionpolypeptide preferably is fused to the C-terminus of a soluble ITAK.General preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990). A genefusion encoding the ITAK:Fc fusion protein is inserted into anappropriate expression vector. ITAK:Fc fusion proteins are allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between Fc polypeptides, yielding divalent ITAK.

Recombinant vectors, including expression vectors, containing a nucleicacid sequence encoding ITAK can be prepared using well known methods.The expression vectors include an ITAK DNA sequence operably linked tosuitable transcriptional or translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are “operably linked”when the regulatory sequence functionally relates to the ITAK DNAsequence. Thus, a promoter nucleotide sequence is operably linked to anITAK DNA sequence if the promoter nucleotide sequence controls thetranscription of the ITAK DNA sequence. The ability to replicate in thedesired host cells, usually conferred by an origin of replication, and aselection gene by which transformants are identified, may additionallybe incorporated into the expression vector.

In addition, sequences encoding appropriate signal peptides that are notnaturally associated. with ITAK can be incorporated into expressionvectors. For example, a DNA sequence for a signal peptide (secretoryleader) may be fused in-frame to the ITAK sequence so that ITAK isinitially translated as a fusion protein comprising the signal peptide.A signal peptide that is functional in the intended host cells enhancesextracellular secretion of the ITAK polypeptide. The signal peptide maybe cleaved from the ITAK polypeptide upon secretion of ITAK from thecell.

Suitable host cells for expression of ITAK polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y. (1985). Cell-freetranslation systems could also be employed to produce ITAK polypeptidesusing RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, an ITAK polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant ITAK polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. To construct an expression vector using pBR322, anappropriate promoter and an ITAK DNA sequence are inserted into thepBR322 vector. Other commercially available vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) andpPLc28 (resident in E. coli RR1 (ATCC 53082)).

ITAK polypeptides alternatively may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia, K. lactis or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657 or in Fleer et.al., Gene 107:285-195 (1991); and van den Berg et. al., Bio/Technology8:135-139 (1990). Another alternative is the glucose-repressible ADH2promoter described by Russell et al. (J. Biol. Chem. 258:2674, 1982) andBeier et al. (Nature 300:724, 1982). Shuttle vectors replicable in bothyeast and E. coli may be constructed by inserting DNA sequences frompBR322 for selection and replication in E. coli (Amp^(r) gene and originof replication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof an ITAK polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad.Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Otherleader sequences suitable for facilitating secretion of recombinantpolypeptides from yeast hosts are known to those of skill in the art. Aleader sequence may be modified near its 3′ end to contain one or morerestriction sites. This will facilitate fusion of the leader sequence tothe structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant ITAK polypeptides. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10:2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

Exemplary expression vectors for use in mammalian host cells can beconstructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991. The vectors may be derived from retroviruses. In place ofthe native signal sequence, a heterologous signal sequence may be added,such as the signal sequence for IL-7 described in U.S. Pat. No.4,965,195; the signal sequence for IL-2 receptor described in Cosman etal., Nature 312:768 (1984); the IL4 signal peptide described in EP367,566; the type I IL-1 receptor signal peptide described in U.S. Pat.No. 4,968,607; and the type II IL-1 receptor signal peptide described inEP 460,846.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express ITAK asa secreted polypeptide in order to simplify purification. Secretedrecombinant polypeptide from a yeast host cell fermentation can bepurified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 296:171, 1984). Urdal et al. describe two sequential,reversed-phase HPLC steps for purification of recombinant human IL-2 ona preparative HPLC column.

As noted above, the present invention provides methods of detectingagonists or antagonists of ITAK, IL-1 and/or TNF-α. As used herein, “anITAK agonist” does not include IL-1 or TNF-α. Such methods permitidentification of elements of the signal transduction pathways of eachof IL-1 and TNF-α.

In one embodiment, the invention thus generally provides a method foridentifying gene products that associate with ITAK, comprising: (a)introducing nucleic acid sequences encoding an ITAK polypeptide into afirst expression vector such that ITAK sequences are expressed as partof a fusion protein comprising a functionally incomplete first portionof a protein that is essential to the viability of a host cell; (b)introducing nucleic acid sequences encoding a plurality of candidategene products that interact or associate with ITAK into a secondexpression vector such that any candidate gene products are expressed aspart of a fusion protein comprising a second functionally incompleteportion of the protein that is essential to the viability of the hostcell; (c) introducing the first and second expression vectors into ahost cell under conditions and for a time sufficient such that host cellsurvival is dependent upon reconstitution of both the first and secondfunctionally incomplete portions of the protein (that is essential tothe viability of the host cell) into a functionally complete protein;and (d) identifying the nucleic acid sequences encoding the candidategene products that associate with ITAK in the second expression vector.

For example, the yeast two-hybrid system (Fields and Song, Nature340:245 (1989); U.S. Pat. No. 5,283,173 to Fields et al.) can be used todetect interactions between ITAK and other proteins or between ITAK andselected compounds, or pools of compounds, that are suspected ofincreasing or decreasing the activity of ITAK, IL-1 and/or TNF-α or ofotherwise employing ITAK to transduce a biological signal. Suchinteractions can be detected by screening for functional reconstitutionof a yeast transcription factor.

Briefly, the yeast two hybrid system was developed as a way to testwhether two proteins associate or interact directly with each other andwas then modified to serve as a method to “capture” candidate proteinsthat interact with a known protein of interest or “bait.” The baitprotein is expressed as a fusion protein with the DNA-binding domain ofGAL4, a yeast transcription factor, in a specially designed yeast strain(Y190) containing reporter genes under GAL4 control. (Durfee et al,Genes & Devel. 7:555, 1993. ) GAL4 is a modular yeast transcriptionfactor with the DNA binding domain confined to the N-terminal 147residues while the transcriptional activation function resides entirelyin the C-terminal 114 residues. Libraries used in the two-hybrid systemhave clones expressing GAL4 activation domain fusion proteins. Themethod detects the reconstitution of GAL4 function when two fusionproteins encode proteins that associate with each other, so that theDNA-binding domain fusion recruits the activation domain fusion intoposition at the GAL4 promoter, leading to transcriptional activation ofthe GAL4-controlled reporter genes.

The ITAK nucleic acid sequences disclosed herein can be cloned into asuitable vector carrying the DNA-binding domain of GAL4 and transformedinto an appropriate yeast strain to produce yeast cells which express aGAL4 DNA-binding domain/ ITAK region fusion protein using methods wellknown in the art. Activation domain cDNA libraries can then be screenedin appropriate vectors. A positive signal in such a two-hybrid assay canresult from cDNA clones that encode proteins that specifically associatewith ITAK such as substrates or activators of ITAK. Knowledge ofproteins that associate with ITAK can also permit searching forinhibitors of IL-1 and/or TNF-α signaling.

The functional interaction between ITAK and its associating proteinsalso permits screening for small molecules that interfere with theITAK/substrate or ITAK/activator association and thereby inhibit IL-1 orTNF-α activity. For example, the yeast two-hybrid system can be used toscreen for IL-1 and/or TNF-α inhibitors as follows. ITAK andactivator/substrate, or portions thereof responsible for theirinteraction, can be fused to the GAL4 DNA binding domain and GAL4transcriptional activation domain, respectively, and introduced into astrain that depends on GAL4 activity for growth on plates lackinghistidine. Compounds that prevent growth can be screened in order toidentify IL-1 and/or TNF-α inhibitors. Alternatively, the screen can bemodified so that ITAK/activator or ITAK/substrate interaction inhibitsgrowth, so that inhibition of the interaction allows growth to occur.Another, in vitro, approach to screening for IL-1 and/or TNF-αinhibition would be to immobilize one of the components, such as ITAK,or portions thereof, in wells of a microtiter plate, and to couple aneasily detected indicator to the other component. An inhibitor of theinteraction is identified by the absence of the detectable indicatorfrom the well.

A high throughput screening assay can also be utilized to identifycompounds that inhibit ITAK activity. For example, natural productextracts, from plant and marine sources, as well as microbialfermentation broths, can be sources of kinase inhibitors and can bescreened for potential ITAK antagonists. Other sources of ITAKantagonists include preexisting or newly generated libraries of smallorganic molecules and preexisting or newly generated combinatorialchemistry libraries. Identification of endogenous ITAK substrate(s), andmapping of their phosphorylation site(s) to determine specificrecognition motif(s), can enable the development of peptide mimeticinhibitors. In addition, in vivo regulation of ITAK activity likelyinvolves endogenous protein inhibitor(s), which can be identified usingthe assay(s) described herein.

These assays also facilitate the identification of other molecules thatinteract with ITAK in a physiologically relevant manner, such asendogenous substrates, activators and the aforementioned natural proteininhibitors. Such molecules include, but are not limited to, receptorsand receptor-associated polypeptides, guanine nucleotide bindingproteins (G proteins), guanine nucleotide exchange factors (GEFs),guanine nucleotide activating proteins (GAPs), transcription activators,and repressors. Additionally, the ITAK assays can serve as readouts toidentify other enzymes involved in a signaling cascade, such as otherkinases, phosphatases and phospholipases.

Accordingly, the invention provides methods of detecting agonists orantagonists of ITAK, IL-1 and/or TNF-α by assaying the downstreamresponse pathway effects of IL-1 or TNF-α signal transduction. In oneaspect of the invention, the method for determining whether a selectedagent is an ITAK agonist comprises (a) exposing the selected agent to anunstimulated ITAK response pathway under conditions and for a timesufficient to allow a stimulation of the pathway; and (b) detectingstimulation of the response pathway and therefrom determining thepresence of an ITAK agonist. In a related aspect, the method fordetermining whether a selected agent is an ITAK agonist comprises (a)measuring the ITAK kinase activity of an ITAK response pathway; (b)exposing the selected agent to the measured ITAK response pathway; and(c) detecting increased ITAK kinase activity in the response pathway.Within another aspect, the invention also provides a method fordetermining whether a selected agent is an ITAK antagonist, comprising:(a) exposing the selected agent to an ITAK response pathway in thepresence of an ITAK agonist under conditions and for a time sufficientto allow a decrease in stimulation of the pathway; and (b) detecting adecrease in the stimulation of the response pathway relative to thestimulation of the response pathway by the ITAK agonist alone, andtherefrom determining the presence of an ITAK antagonist. Such methodsmay include assays of cellular proliferation (Raines et al., Science243:393, 1989), prostaglandin production (Curtis et al., Proc. Nat.Acad. Sci. USA 86:3045, 1989), colony stimulating factor production(Curtis et al., 1989), cell surface immunoglobulin up-regulation (Giriet al., J. Immunol. 131:223, 1984), NFκ-B activation (Shirakawa et al.,Mol Cell. Biol. 9:959, 1989), or other established biological signaltransduction assays known to those skilled in the art.

In a related aspect, ITAK polypeptides according to the invention may beused for the structure-based design of an inhibitor of IL-1 or TNF-αdownstream effects, as well as for the design of ITAK-inhibitors. Suchstructure-based design is also known as “rational drug design.” Suchdesign can include the steps of determining the three-dimensionalstructure of such an ITAK polypeptide, analyzing the three-dimensionalstructure for the likely binding sites of substrates (as well asanalyzing ITAK for electrostatic potential of the molecules, proteinfolding, etc.), which sites represent predictive reactive sites,synthesizing a molecule that incorporates one (or more) predictivereactive site, and determining the ITAK-inhibiting activity of themolecule. (Sudarsanam et al., J. Comput. Aided. Mol. Design 6:223, 1992)ITAK polypeptides can be three-dimensionally analyzed by, for example,X-ray crystallography, nuclear magnetic resonance or homology modeling,all of which are well-known methods. For example, most of the design ofclass-specific inhibitors of metalloproteases has focused on attempts tochelate or bind the catalytic zinc atom. Synthetic inhibitors areusually designed to contain a negatively-charged moiety to which isattached a series of other groups designed to fit the specificitypockets of the particular protease.

Because only the cytokines IL-1 and TNF-α appear to induce ITAKactivity, the present invention offers the advantage of selectivelyblocking functional cellular responses to these cytokines. Thus, ITAKfacilitates the discovery of inhibitors of ITAK, and thus, inhibitors ofthe effects of excessive IL-1 and TNF-α release. This use of ITAK forthe screening of potential inhibitors thereof is important and caneliminate or reduce the possibility of interfering reactions withcontaminants.

Turning to another aspect of the invention, IL-1 activity is initiatedby the IL-1 molecule binding to a membrane-bound IL-1 receptor, which inturn interacts with cytoplasmic proteins associated with the cytoplasmicregion of the IL-1 receptor. For example, the cytoplasmic domain of thetype I IL-1 receptor is associated with a GTP-ase activating protein(GAP) that is referred to herein as IIP1, and which is described ingreater detail in an application entitled “IL-1 Receptor InteractingProtein” (U.S. Ser. No. 08/584,831, filed Jan. 11, 1996). GAP proteins,such as IIP1, interact with G proteins that in turn interact withcytoplasmic effector molecules (e.g., protein kinases or ion channels)that carry out early signaling functions. G-proteins bind to guaninenucleotides (GDP or GTP). When a G protein is bound to GTP it interactswith effector molecules to generate a biological signal (the “ON”configuration). Conversely, when a G-protein is bound to GDP, it doesnot interact and is not capable of generating a biological signal (the“OFF” configuration). G-proteins are in a constant state of equilibriumbetween the GTP-bound and GDP-bound forms. When IL-1 is not bound toIL-1 receptor, IIP1 catalyzes the hydrolysis of GTP to GDP, forcing theequilibrium between the GTP-bound and GDP-bound G-protein towards theGDP-bound “off” form, thereby preventing an IL-1 signal. When IL-1 bindsto IL-1 receptor, the IIP1 interaction with GTP- and GDP-bound forms ofthe G-protein is interrupted, causing the equilibrium to shift towardsthe GTP-bound “on” form, and thereby transmitting an IL-1 signal. Thus,the net effect of IIP1 is to suppress the G-protein-linked signal.

A similar G-protein-regulated signaling pathway may control induction ofITAK activity in response to IL-1 or TNF-α. Conversely, the IL-1 orTNF-α-mediated induction of ITAK activity may control the function ofone or more G-proteins. The disclosure herein of an ITAK polypeptidedomain having pronounced amino acid sequence homology to known guaninenucleotide exchange factors (such as RCC1 (Bischoff and Ponstingl,Nature 354:80, 1991) is compatible with either of these schemes, whichare not mutually exclusive. Thus, the provision herein of ITAK providesalternative avenues for the investigation, detection and possiblecontrol of G-protein-related pathways. ITAK may be used as a reagent toidentify (a) a G protein that regulates—or is regulated by—ITAK andwhich is involved in IL-1 or TNF-α signaling, and (b) other proteinswith which ITAK interacts that would be involved in IL-1 or TNF-α signaltransduction pathways. These other proteins, including the G protein,are then useful tools to search for other inhibitors of IL-1 or TNF-αsignaling. ITAK can also be used by coupling recombinant ITAK protein toan affinity matrix.

In another aspect, the present invention also provides nucleic acidprobes based upon a nucleic acid molecule encoding ITAK. Such probes canbe used in accordance with hybridization and other assays known in theart, for example, to detect ITAK genes in candidate samples, such assamples derived from candidate cell lines or animal strains or species.Such probes preferably specifically hybridize to the ITAK gene underappropriately determined conditions that may be conditions of moderateor high stringency (see, e.g., Sambrook et al., supra), and generallycomprise at least about 15 nucleotides, typically at least about 18nucleotides or at least about 20 nucleotides, and preferably from about18 to about 35 nucleotides and even more preferably several hundrednucleotides. However, such probes can comprise up to an entire ITAKgene, if desired.

The present invention also provides antisense or sense nucleotidescomprising a single-stranded nucleic acid sequence (either RNA or DNA)capable of binding to a target ITAK mRNA sequence (forming a duplex) orto the ITAK sequence in the double-stranded DNA helix (forming a triplehelix). Such nucleotides often comprise a fragment of the coding regionof ITAK cDNA but may also comprise a fragment of the non-coding regionof ITAK cDNA. Typically such nucleotides comprise an ITAK-specificfragment. Such a fragment generally comprises at least about 15nucleotides, typically at least about 18 nucleotides or at least about20 nucleotides, and preferably from about 18 to about 35 nucleotides andeven more preferably several hundred nucleotides. The ability to createan antisense or a sense nucleotide, based upon a cDNA sequence for agiven protein, is described in, for example, Stein and Cohen, CancerRes. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense nucleotides to target nucleic acidsequences results in the formation of complexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense nucleotides thus may beused to block expression of ITAK proteins. Blockade of ITAK expressionin this manner can be useful therapeutically in inflammatory diseasesituations. Antisense or sense nucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense nucleotides include thoseoligonucleotides that are covalently linked to organic moieties, such asthose described in WO 90/10448, and other moieties that increaseaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense nucleotides to modify binding specificities of theantisense or sense nucleotide for the target nucleotide sequence.

Antisense or sense nucleotides may be introduced into a cell containingthe target nucleic acid sequence by any gene transfer method, including,for example, CaPO₄-mediated DNA transfection, electroporation, or byusing gene transfer vectors such as Epstein-Barr virus. Antisense orsense nucleotides are preferably introduced into a cell containing thetarget nucleic acid sequence by insertion of the antisense or sensenucleotide into a suitable retroviral vector, then contacting the cellwith the retrovirus vector containing the inserted sequence, either invivo or ex vivo. Suitable retroviral vectors include, but are notlimited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived fromM-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C(see PCT Application US 90/02656).

Sense or antisense nucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense nucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense nucleotide may be introduced intoa cell containing the target nucleic acid sequence by formation of anucleotide-lipid complex, as described in WO 90/10448. The sense orantisense nucleotide-lipid complex is preferably dissociated within thecell by an endogenous lipase.

In another aspect, the present invention provides binding partners thatspecifically interact with ITAK. Such binding partners, typicallyantibodies, can be useful for inhibiting IL-1 or TNF-α activity in vivoand for detecting the presence of ITAK in a sample. SuitableITAK-binding partners include antibodies that are immunoreactive withITAK, and preferably monoclonal antibodies against ITAK, and otherproteins that are capable of high-affinity binding to ITAK. The term“antibodies” includes polyclonal antibodies, monoclonal antibodies,fragments thereof such as F(ab′)₂, and Fab fragments, as well as anyrecombinantly produced binding partners. Antibodies are defined to bespecifically binding if they bind ITAK with a K_(a) of greater than orequal to about 10⁷ M⁻¹. Affinities of binding partners or antibodies canbe readily determined using conventional techniques, for example thosedescribed by Scatchard et al., Ann. N.Y. Acad. Sci. 51:660 (1949).Determination of other proteins as binding partners of ITAK can beperformed using, for example, the yeast two-hybrid screening systemdescribed herein. The present invention also includes the use of ITAK,and peptides based on the amino acid sequence of ITAK, to preparebinding partners and antibodies that specifically bind to ITAK.

ITAK binding partners that are polyclonal antibodies can be readilygenerated from a variety of sources, for example, horses, cows, goats,sheep, dogs, chickens, rabbits, mice or rats, using procedures that arewell-known in the art. In general, purified ITAK, or a peptide based onthe amino acid sequence of ITAK that is appropriately conjugated, isadministered to the host animal typically through parenteral injection.The immunogenicity of ITAK may be enhanced through the use of anadjuvant, for example, Freund's complete or incomplete adjuvant.Following booster immunizations, small samples of serum are collectedand tested for reactivity to ITAK or the ITAK peptides. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988, as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radio-immunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

By virtue of the isolated ITAK provided herein, monoclonal antibodiesspecific for ITAK are readily prepared using well-known procedures, seefor example, the procedures described in U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: ANew Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,and Bechtol (eds.), 1980; Harlow, supra. Briefly, host animals such asmice are injected intraperitoneally at least once, and preferably atleast twice at about 3 week intervals with isolated and purified ITAK orconjugated ITAK peptide, optionally in the presence of adjuvant. Mousesera are then assayed by conventional dot blot technique or antibodycapture (ABC) to determine which animal is best to fuse. Approximatelytwo to three weeks later, the mice are given an intravenous boost ofITAK or conjugated ITAK peptide. Mice are later sacrificed and spleencells fused with commercially available myeloma cells, such as Ag8.653(ATCC), following established protocols. Briefly, the myeloma cells arewashed several times in media and fused to mouse spleen cells at a ratioof about three spleen cells to one myeloma cell. The fusing agent can beany suitable agent used in the art, for example, polyethylene glycol(PEG). Fusion is plated out into plates containing media that allows forthe selective growth of the fused cells. The fused cells can then beallowed to grow for approximately eight days. Supernatants fromresultant hybridomas are collected and added to a plate that is firstcoated with goat anti-mouse Ig. Following washes, a label, such as,¹²⁵I-ITAK is added to each well followed by incubation. Positive wellscan be subsequently detected by autoradiography. Positive clones can begrown in bulk culture and supernatants are subsequently purified over aProtein A column (Pharmacia).

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas,” Strategies in Molecular Biology 3:1-9 (1990). Similarly,binding partners can be constructed using recombinant DNA techniques toincorporate the variable regions of a gene that encodes a specificbinding antibody. Such a technique is described in Larrick et al.,Biotechnology 7:394 (1989).

Other types of “antibodies” may be produced using the informationprovided herein in conjunction with the state of knowledge in the art.For example, antibodies that have been engineered to contain elements ofhuman antibodies that are capable of specifically binding ITAK are alsoencompassed by the invention.

Once isolated and purified, ITAK binding partners can be used to detectthe presence of ITAK in a sample using established assay protocols.Further, the binding partners, typically the antibodies, of theinvention may be used therapeutically to bind to ITAK and inhibit itsactivity in vivo. Such ITAK-binding partners can be bound to a solidphase such as a column chromatography matrix or a similar substratesuitable for identifying, separating or purifying molecular componentsobtained from cells that express ITAK. Adherence of ITAK or ITAK-bindingproteins to a solid phase contacting surface can be accomplished by anymeans, for example, magnetic microspheres can be coated withITAK-binding proteins and held in the incubation vessel through amagnetic field. Cell extracts are contacted with the solid phase thathas ITAK or ITAK-binding proteins thereon. ITAK or ITAK-associatedspecies bind to the fixed-binding protein and unbound material is thenwashed away. This affinity-binding method is useful for purifying,screening or separating such ITAK-associated molecules from solution.Methods of releasing positively selected components from the solid phaseare known in the art and encompass, for example, the use of pH changes,altered salt concentration, or chaotropic agents.

ITAK or a fragment or variant thereof can also be useful itself as atherapeutic agent in inhibiting IL-1 and/or TNF-α signaling. ITAKagonists or ITAK antagonists provided by the invention are also usefulas therapeutic agents in inhibiting IL-1 and/or TNF-α signaling, aloneor in combination with ITAK or a fragment thereof or a variant thereof.ITAK, or an ITAK agonist or an ITAK antagonist, is introduced into theintracellular environment by well-known means, such as by encasing ITAK(or its agonist or antagonist) in liposomes or coupling it to amonoclonal antibody targeted to a specific cell type.

When used as a therapeutic agent, ITAK, an ITAK agonist, or an ITAKantagonist can be formulated into pharmaceutical compositions accordingto known methods. In a preferred embodiment, the ITAK contains amutation of the lysine residue at position 81 to another amino acid, forexample the ITAK variant known as ITAK A81, which contains a mutationfrom lysine to alanine. ITAK, an ITAK agonist, or an ITAK antagonist canbe introduced into the intracellular environment using methods wellknown in the field, such as encasing ITAK in liposomes or coupling ITAKto a monoclonal antibody targeted to a specific cell type.

ITAK, an ITAK agonist, or an ITAK antagonist can be combined inadmixture, either as the sole active material or with other known activematerials, with pharmaceutically suitable diluents (e.g., Tris-HCl,acetate, phosphate), preservatives (e.g., Thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants and/or carriers.Suitable carriers and their formulations are described in Remington'sPharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition,such compositions can contain ITAK, or an ITAK agonist or an ITAKantagonist, complexed with polyethylene glycol (PEG), metal ions, orincorporated into polymeric compounds such as polyacetic acid,polyglycolic acid, hydrogels, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Such compositions will influence thephysical state, solubility, stability, rate of in vivo release, and rateof in vivo clearance of ITAK.

Compositions of the invention that are nucleotide sequences encodingITAK or a fragment thereof, an ITAK variant or a fragment thereof, anITAK agonist or a fragment thereof, or an ITAK antagonist or a fragmentthereof, are used as therapeutic agents according to gene therapystrategies. Generally such nucleotide sequences are incorporated intovectors leading to expression of the desired nucleotide sequences; suchvectors are readily constructed by those skilled in the art. Inaddition, administration of such vectors by various means is well knownto those skilled in the art.

Such vectors for gene therapy may be retroviral vector constructs or maybe developed and utilized with other viral carriers including, forexample, poliovirus (Evans et al., Nature 339:385-388, 1989; and Sabin,J. Biol. Standardization 1:115-118, 1973); rhinovirus; pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS86:317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989;Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112 and4,769,330; WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114,1979); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989;McMichael et al., N. Eng. J. Med. 309:13-17, 1983; and Yap et al.,Nature 273:238-239, 1978); adenovirus (Berkner, Biotechniques 6:616-627,1988; Rosenfeld et al, Science 252:431-434, 1991); parvovirus such asadeno-associated virus (Samulski et al., J. Vir. 63:3822-3828, 1989;Mendelson et al, Virol. 166:154-165, 1988); and herpes (Kit, Adv. Exp.Med. Biol. 215:219-236, 1989).

Once a vector has been prepared, it may be therapeutically administeredto a warm-blooded animal. As noted above, methods for administering avector are well known to those skilled in the art and include, forexample, by direct administration, or via transfection utilizing variousphysical methods, such as lipofection (Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417, 1989), direct DNA injection (Acsadi et al.,Nature 352:815-818, 1991); microprojectile bombardment (Williams et al,PNAS 88:2726-2730, 1991); liposomes (Wang et al., PNAS 84:7851-7855,1987); CaPO₄ (Dubensky et al., PNAS 81:7529-7533, 1984); or DNA ligand(Wu et al., J. Biol. Chem. 264:16985-16987, 1989).

Pharmaceutical compositions for gene therapy comprising one of theabove-described recombinant viruses containing nucleotide sequencesencoding ITAK or a fragment thereof, an ITAK variant or fragmentthereof, an ITAK agonist or a fragment thereof, or an ITAK antagonist orfragment thereof are provided. The composition may be prepared either asa liquid solution, or as a solid form (e.g., lyophilized) which issuspended in a solution prior to administration. In addition, thecomposition may be prepared with suitable carriers or diluents foreither injection, oral, or rectal administration. Generally, therecombinant virus is utilized at a concentration ranging from 0.25% to25%, and preferably about 5% to 20% before formulation. Subsequently,after preparation of the composition, the recombinant virus willconstitute about 1 μg of material per dose, with about 10 times thisamount material (10 μg) as copurified contaminants. Preferably, thecomposition is prepared in 0.1-1.0 ml of aqueous solution formulated asdescribed below.

Pharmaceutically acceptable carriers or diluents are nontoxic torecipients at the dosages and concentrations employed. Representativeexamples of carriers or diluents for injectable solutions include water,isotonic saline solutions which are preferably buffered at aphysiological pH (such as phosphate-buffered saline or Tris-bufferedsaline), mannitol, dextrose, glycerol, and ethanol, as well aspolypeptides or proteins such as human serum albumin. A particularlypreferred composition comprises a vector or recombinant virus in 10mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2 and 150 mM NaCl. In thiscase, since the recombinant vector represents approximately 1 μg ofmaterial, it may be less than 1% of high molecular weight material, andless than {fraction (1/100,000)} of the total material (includingwater). This composition is stable at −70° C. for at least six months.The composition may be injected intravenously (i.v.), subcutaneously(s.c.), or intramuscularly (i.m.). Oral formulations may also beemployed with carriers of diluents such as cellulose, lactose, mannitol,poly (DL-lactide-co-glycolate) spheres, and/or carbohydrates such asstarch. The composition may take the form of, for example, a tablet, gelcapsule, pill, solution, or suspension, and additionally may beformulated for sustained release. For rectal administration, preparationof a suppository may be accomplished with traditional carriers such aspolyalkalene glucose, or a triglyceride.

The following Examples provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention which is set forth in the appended claims. In the followingExamples, all methods described are conventional unless otherwisespecified.

EXAMPLES Example 1 Determination of ITAK Activity

Cloned ITAK gene sequences were initially detected by comparativeanalysis of the partial amino acid sequence of an isolated and purifiedrabbit IL-1-induced β-casein kinase polypeptide, IL-1/TNF-α-activatedkinase (ITAK). Cloned human nucleotide sequences were identified thatencoded polypeptide regions characteristic of protein kinases and thatdisplayed amino acid sequence homology with ITAK-derived peptides.

ITAK activity was originally detected by its ability to phosphorylateintact β-casein (Guesdon et al., J. Biol. Chem. 268:4236 (1993); Guesdonet al., Biochem. J. 304:761 (1994)). However the assay utilizedthroughout this purification is a second generation peptide-based assay.Three sites (Ser 57, Ser 124, and Ser 142) of ITAK-mediatedphosphorylation of β-casein were identified using methods known in theart. Dephosphorylated bovine β-casein was then ³²P-labeled with ITAKunder previously described conditions (Guesdon et al., 1993; Guesdon etal., 1994; supra). Radiolabeled β-casein was proteinase digested and theresultant peptides were separated by two-dimensional thin layerchromatography and/or reverse phase high performance liquidchromatography (RP-HPLC). Isolated, radioactive peptides were thensequenced to determine the amino acid sequences of phosphorylationacceptor sites. Three peptides containing phosphoserine were identifiedin this manner. Various peptide substrates composed of sequences aroundand including these three serine residues were synthesized on an ABIModel 430 Peptide Synthesizer. All peptide substrates were synthesizedhaving multiple N-terminal or C-terminal basic residues to mediatepeptide binding to phosphocellulose filters; this is a commonly usedapproach in peptide-based kinase assays. (Glass et al., Anal. Biochem.87:566,1978; Casnellie et al., Proc. Nat. Acad. Sci. USA 79:282, 1982.)Kinetic analysis of the various potential peptide substrates resulted inselection of the following peptide for the standard ITAK assay:

-   -   Arg-Arg-Arg-His-Leu-Pro-Pro-Leu-Leu-Leu-Gln-Ser-Trp-Met-His-Gln-Pro-His-Gln        (single letter code: RRRHLPPLLLQSWMHQPHQ) (SEQ ID:NO 3)

In the standard ITAK assay, 10 μl of ITAK-containing sample is added to10 μl of 2× assay buffer (40 mM Hepes, pH 7.4/20 mM MnCl₂/20 μM ATP/1μCi γ-(³²P)-ATP) containing 2 mM peptide substrate, the reactionproceeds for 20 minutes at 30° C., then is stopped by adding 10 μl offormic acid. Blank controls consist of assays performed in the absenceof peptide substrate. After reactions are stopped, assay mixtures arespotted onto circular 2.5 cm phosphocellulose filters (P81, Whatman,Fairfield, N.J.), washed twice with 75 mM H₃PO₄, and placed in 20 mlborosilicate scintillation vials for Cerenkov counting in a β-counter.Net counts per minute, cpm, (sample cpm minus blank cpm) are used tocalculate picomoles of phosphate incorporated into the peptidesubstrate. One Unit of ITAK activity is defined as that amount of ITAKnecessary to incorporate 1 picomole of phosphate into the peptidesubstrate, RRRHLPPLLLQSWMHQPHQ (SEQ ID:NO 3), in one minute understandard assay conditions. Specific activity is defined as Units of ITAKactivity per milligram of protein. Adaptations of this assay for largescale screening may include use of biotinylated ITAK substrate peptidein a scintillation proximity assay (SPA) with streptavidin coated SPAbeads, or covalent modification of the ITAK substrate peptide withfluorescent tags by techniques known in the art.

Example 2 Purification of ITAK

This example describes the purification of IL-1-induced, rabbit lungITAK in quantities sufficient to permit partial amino acid sequencing ofthe ITAK protein (see Table 1). Rabbit lungs were chosen because theydisplayed the greatest increase in ITAK activity in response to IL-1α,when compared with untreated control animal tissues. This exampledetails the purification of ITAK from 70 pairs of lungs taken fromIL-1α-treated rabbits.

Briefly, New Zealand White rabbits (2.0-2.5 kg) were intravenously earinjected with 100 μg of human recombinant IL-1α/kg body weight in atotal volume of 0.5 ml in PBS (phosphate buffered saline). Fifteenminutes after injection animals were sacrificed by cervical dislocationand the lungs rapidly excised (within 2-3 min). Once removed, lungs werefast-frozen on dry ice, then stored at −80° C.

Lungs were cut into small pieces (˜0.5 cm³) while the tissue was stillpartially frozen, in ice-cold Wash Buffer (PBS containing proteinaseinhibitors, 0.1 mM leupeptin and 1.0 mM phenylmethylsulfonyl fluoride(PMSF)) on ice. Minced lungs were washed at least twice, to removecontaminating blood proteins, with 100 ml of ice cold Wash Buffer/pairof lungs. Generally five pairs of lungs were processed at a time;therefore each wash was performed in 500 ml of Wash Buffer, for tenminutes with constant agitation, after which buffer was removed bydecanting and aspiration.

Following the second wash, minced lung tissue was immediately placed inice-cold Homogenization Buffer (HB: 25 mM Tris-HCl, pH 7.5/100 mMβ-glycerophosphate/25 mM para-nitrophenyl phosphate/10 mM sodiumorthovanadate/2 mM DTT (dithiothreitol)/1 mM MgCl₂/5 mM EDTA(Ethylenediaminetetraacetic Acid)/5 mM EGTA (EthyleneGlycol-bis(β-aminoethyl) Ether) N,N,N′,N′-Tetraacetic Acid)/5 mMbenzamidine/1 μM E-64(trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane)/1 mM PMSF/0.1 mMleupeptin), and further minced. Minced lungs were homogenized at a finalratio of 10:1 (vol. HB (ml): mass tissue (gm)). Initially, the mincedlungs were homogenized in 75% of the total volume of HB, solid materialwas pelleted by centrifugation at 12,000 rpm for 30 min at 4° C. and thepellets re-homogenized in the remaining 25% of the buffer.

Homogenization (of the minced tissue in 75% of the buffer) was performedusing a Brinkman Homogenizer at setting #8 for two 20 second pulses.Solid material was removed by centrifugation at 12,000 rpm for 30minutes at 4° C. The supernatant was removed, pellets resuspended in theremaining 25% of the HB and re-homogenized at setting #8 for 30 seconds.Another centrifugation at 12,000 rpm for 30 minutes at 4° C. was used toremove insoluble material. Both supernatants were combined and furtherclarified by gravity filtration through glass wool.

This preparation, which utilized 70 pairs of lungs (wet weight 560grams) isolated from rHuIL-1α-treated rabbits, yielded 5.7 liters oflung homogenate. The lung homogenate was made 25% with respect toammonium sulfate by the gradual addition of 764 gm of solid ammoniumsulfate with constant, slow stirring at 4° C. Once all of the ammoniumsulfate was in solution, stirring was stopped and the homogenateincubated at 4° C. overnight. The 0-25% ammonium sulfate precipitate wascollected by centrifugation at 12,000 rpm for 30 min at 4° C. Pelletedprecipitates were resolubilized, in four equal batches, in 500 ml eachof Buffer A (20 mM Tris, pH 8.5/50 mM β-glycerophosphate/2 mM DTT/1 mMEDTA/1 mM EGTA/0.1 mM PMSF/0.1 mM leupeptin). The resolubilized 0-25%precipitate was dialyzed against two changes (10 liters each) of BufferA at 4° C. overnight. After dialysis, residual insoluble material wasremoved by centrifugation at 20,000 rpm for 30 min at 4° C. Theresultant supernatant was sequentially filtered through a glass fiberpre-filter, a 0.8 μm filter, and finally a 0.45 μm filter (Corning,Corning, N.Y.).

Buffers used for all chromatography were filtered through 0.45 μmfilters (Corning) prior to use. Each of the four filtered batches(containing 550-600 ml) was individually applied to a 25 ml (10.5×1.6cm) column of Source 15Q (Pharmacia, Piscataway, N.J.) previouslyequilibrated with Buffer A, at a flow rate of 6.0 ml/min. The column wasthen washed with ten bed volumes (250 ml) of Buffer A at 6.0 ml/min.Bound protein was eluted with an increasing linear gradient of NaCl(0-0.5 M) in Buffer A at 6.0 ml/min over a period of 56.6 minutes. Fourand a half ml fractions were collected and ten μl from each fractionassayed for ITAK activity. All chromatographic steps were performed at4° C., unless otherwise indicated.

ITAK activity eluted from Source 15Q at a NaCl concentration of 200-300mM (Table 1). Fractions containing eluted ITAK activity from the fourseparate Source 15Q runs were pooled, diluted 1:2 with Buffer B (BufferA containing 10% glycerol) and applied to a 50 ml (9.5×2.6 cm) column ofReactive Green 19 (Sigma, St. Louis, Mo.), previously equilibrated withBuffer B, at a flow rate of 2.5 m/min. After loading, the column waswashed with four bed volumes (200 ml) of Buffer B at 2.5 ml/min. Proteinwas eluted from the Green 19 column with an increasing linear gradientof NaCl (0-2.0 M) in Buffer B at 2.5 ml/min over 80 min. Four-mlfractions were collected and 5 μl aliquots from each fraction assayedfor ITAK activity. ITAK activity eluted in a broad peak with a NaClconcentration of from 1.0-1.5 M. Active fractions were pooled andconcentrated in a Centriprep 30 concentrator (Amicon, Beverly, Mass.) toa final volume of 5.0 ml.

The ITAK concentrate was loaded onto a HiLoad 26/60 Superdex 200 sizeexclusion chromatography column (Pharmacia, Piscataway, N.J.) previouslyequilibrated with Buffer B. Protein was eluted with Buffer B at 2.5ml/min. Fractions of 4.0 ml were collected and 5 μl aliquots assayed forITAK activity. Gel filtration calibration standards (BioRad, Hercules,Calif.) chromatographed under identical conditions were used to estimatethe apparent molecular weight of ITAK; its elution was consistent with aMr˜350 kD.

The pooled peak fractions of ITAK activity eluted from Superdex 200 weremade 0.1% in NP-40 by addition of the appropriate amount of a 10% NP-40solution (Pierce, Rockford, Ill.), incubated at 37° C. for 5 minutes,then immediately applied onto a 25 ml (12.5×1.6 cm) column ofHeparin-Sepharose (Pharmacia) previously equilibrated with Buffer C(Buffer B containing 0.1% NP-40), at a flow rate of 2.0 m/min. This stepand all subsequent chromatographic steps were conducted at roomtemperature (20° C.). After loading, the column was washed with fourcolumn volumes (100 ml) of Buffer C at the same flow rate. The columnwas developed with an increasing linear gradient of NaCl (0-1.0 M) inBuffer C at 2.0 ml/min over 50 min. One ml fractions were collectedthroughout the salt gradient and 2 μl from each fraction assayed forITAK activity. ITAK eluted in the 175-250 mM NaCl region of thegradient.

Active fractions were combined and diluted 1:5 with Buffer C pH adjustedto 8.0, incubated at 37° C. for 5 minutes, then immediately applied to a1.0 ml HR5/5 MonoQ column (Pharmacia), previously equilibrated withBuffer C pH 8.0, at 1.0 ml/min. The column was washed at 1.0 m/min with10 column volumes (10 ml) of Buffer C pH 8.0, before being developedwith an increasing linear gradient of NaCl (0-0.5 M) in Buffer C pH 8.0,also at 1.0 ml/min for 20 minutes. Fractions of 0.5 ml were collectedthroughout the salt gradient and 1 μl from each assayed for ITAKactivity (Table 1). ITAK activity eluted as a single, well-resolved peakin the 200-250 mM NaCl portion of the gradient An additional 1 μl wasremoved from each fraction for analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), using pre-cast 8-16%Novex (San Diego, Calif.) gradient gels.

Prior to SDS-PAGE the 1 μl ITAK-containing fractions from the MonoQcolumn were incubated under modified kinase assay conditions (20 mMHepes-pH 7.4/10 mM MnCl₂/10 μM ATP/0.5 μCi γ-(³²P)-ATP, for 45 min at30° C.) in the absence of exogeneously added substrate. This procedurehad previously resulted in the ³²P-labeling of endogeneous moietiesestimated to be ˜110-125 kD. Following electrophoresis the gel wassilver stained and the radiolabeled band(s) identified using aPhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Prominent silverstained bands estimated to be 90, 100 and 110 kD were observed tocorrespond with ITAK activity. The 110 kD band and two poorly stainedbands at 120 and 125 kD migrated to positions on the gel coinciding withthe ³²P-labeled moieties. All molecular weight estimates were based ondirect comparison with Novex Wide Range Protein Standardselectrophoresed on the same gel.

Eluted MonoQ column fractions having ITAK activity were combined, pHadjusted to 7.0 with 2.0 M Tris-HCl, pH 7.0, then applied in fourseparate batches (300-400 μl each) to a 60×0.75 cm Bio-Sil SEC-400 HPLCgel filtration column (BioRad, Hercules, Calif.) previously equilibratedwith Buffer D (20 mM Tris-HCl, pH 7.0/10 mM β-glycerophosphate/1 mMDTT/1 mM EDTA/1 mM EGTA/1 mM PMSF/0.1 mM leupeptin/10% glycerol/0.1%NP-40). Proteins were eluted from the column at a flow rate of 0.5m/min, 0.5 ml fractions were collected, and 0.5 μl from each fractionassayed for ITAK activity. An additional 0.5 μl was used for ³²Pradiolabeling with γ-(³²P)-ATP under modified kinase assay conditionswithout exogenous substrate, SDS-PAGE and silver staining as describedabove. Again the 90 and 100 kD bands co-eluted with ITAK activity, asdid the endogenously ³²P-labeled 110, 120 and 125 kD bands. Gelfiltration calibration standards (BioRad) were chromatographed underidentical conditions immediately after the final ITAK run. ITAK(Table 1) eluted from the Bio-Sil SEC-400 column (all four runs) at anelution volume consistent with a M,=350 kD.

ITAK-containing fractions from the HPLC gel filtration column werecombined and applied to a 35 μl (5×0.1 cm) microbore MonoQ (Pharmacia)column previously equilibrated with Buffer E (20 mM Tris-HCl, pH 8.5/10mM β-glycerophosphate/1 mM DTT/1 mM EDTA/1 mM EGTA/1 mM PMSF/ 0.1 mMleupeptin/10% glycerol/0.1% NP-40). ITAK was applied to the column at 50μ/min; multiple loadings were necessary and in each case the column waswashed with Buffer E until the absorbance at 280 nm returned tobaseline. After the final loading the column was washed with anadditional 30 column volumes of Buffer E. All loadings and washings wereperformed at a flow rate of 50 μl/min. Protein was eluted from thecolumn with a steep increasing linear gradient of NaCl (0-0.5 M) inBuffer E at a flow rate of 50 μl/min over a period of 10 min. Fractionsof 50 μl were collected and 0.25 μl was removed from each fraction toassay for ITAK activity. An additional 0.25 μl was removed for ³²Pradiolabeling with γ-(³²P)-ATP under modified kinase assay conditionswithout exogenous substrate, SDS-PAGE and silver staining as detailedabove.

Virtually all (>95%) of the ITAK activity eluted in a single fraction(Fraction #12, Table 1) which contained the previously observedunlabeled 90 and 100 kD bands as well as the (³²P)-labeled 110, 120 and125 kD bands. Approximately one third of the ITAK containing fractionwas used for preparative gel electrophoresis. The sample was firstendogenously labeled with ³²P as described above, after which it wasreduced (with excess DTT at 100° C. for 30 min) and then alkylated withan excess of iodoacetamide for 15 minutes in the dark. Electrophoresiswas performed using 8-16% pre-cast Novex gradient gels and run at 100 V(constant voltage) for 30 min, then at 150 V (constant voltage) for anadditional 90 min. Following electrophoresis the gel was stained withCoomassie Brilliant Blue G-250, destained, Saran-wrapped and exposed toa Storage Phosphor Screen for Phosphorlmager identification of theradiolabeled band(s). Those bands co-purifying with ITAK activity,including the radiolabeled bands, were excised from the gel for in-geltrypsin digestion using a modification of techniques known in the art.(Henzel et al., in Methods: A Companion to Methods in Enzymology 6, pp.239-247, 1994. )

Three slices excised from radioactive regions of the gel were estimatedto contain proteins having molecular masses of 110, 120 and 125 kD,based on comparison with co-electrophoresed Wide Range Protein Standards(Novex, San Diego, Calif.). These gel slices were Cerenkov counted andcontained 3.0×10⁵, 6.9×10⁵ and 8.3×10⁵ cpm, respectively. In-gel trypsindigestion was performed on these gel slices using sequencing gradetrypsin (Promega, Madison, Wisc.) at a 1:10 (w/w) ratio. Digestion wasperformed in 20 mM NH₄HCO₃, pH 8.0 at 37° C. for 16 hours. The resultantpeptides were isolated from the gel bits by extraction with 60%acetonitrile/5% formic acid using both incubation at 37° C. andsonication to facilitate recovery of peptides.

Recovered peptides were briefly vacuum concentrated (Speed-Vac SC 100,Savant, Farmingdale, N.Y.) to remove the majority of acetonitrile, thenseparated by applying the material to a capillary C₁₅ (Vydac, Hesperia,Calif.) column previously equilibrated with 0.1% trifluoroacetic acid(TFA) at a flow rate of 15 μl/min. After loading, the capillary columnwas exhaustively washed with 0.1% TFA at 15 μl/min. Peptides were elutedwith an ascending gradient of acetonitrile (0-90%, 1.0% per minute) over90 min. Eluted peptides were monitored spectrophotometrically at 214 nmand fractions hand collected. Tryptic peptide maps of the 110 and 120 kDbands were virtually identical and the map of the 125 kD band wassimilar though less well defined, suggesting that these three bands werelikely modified forms of the same protein. A small portion of eachfraction (3-5%) was analyzed by MALDI (matrix-assisted laser desorptionmass spectroscopy) using a Lasermat Mass Analyzer (Finnigan MAT) and/orby triple quadrapole mass spectroscopy (Finnigan MAT TSQ 700 withelectrospray ionization), and the remainder was sequenced by Edmandegradation using either an ABI 476A or an ABI 494 automated proteinsequencer. These further analyses of peptides derived from the threeradiolabeled bands corroborated the hypothesis that the 110, 120 and 125kD bands are related.

ITAK was found to contain the following sequences:

(SEQ ID:NO 4) Gly-Ala-Phe-Gly-Glu-Ala-Thr-Leu-Tyr-Arg (SEQ ID:NO 5)Val-Thr-Leu-Leu-Asn-Ala-Pro-Thr-Lys

Based on homology to other kinases, the sequence depicted as SEQ ID:NO 4resembles a truncated version of a kinase signature motif. (Hanks etal., Science 241:42, 1988. ) The presence of this rabbit ITAK sequencefragment, which identified this molecule as a kinase, permittedcomparison to the sequences of cDNA clones, derived from independentbiological source materials, that also contained kinase motifs (SeeExamples 3, 4). The translation of one such partial clone, generatedfrom a subtracted human dendritic cell cDNA library and called HH0381,revealed that this cDNA clone contained sequences identical to SEQ ID:NO4. An extended version of the HH0381 cDNA called clone 7 was found tocontain human cDNA-derived nucleotide sequences that translated to bothof the amino acid sequences shown in SEQ ID:NOS 4 and 5, which wereidentified in purified rabbit ITAK peptides. In addition, other aminoacid sequences present among the tryptic peptides generated frompurified rabbit lung 110, 120 and 125 kD ITAK were also found encoded byclone 7. These shared sequences are depicted here as SEQ ID:NO 6 and SEQID:NO 7.

Ser-Ser-Thr-Val-Thr-Glu-Ala-Pro-Ile-Ala-Val-Val-Thr-Ser-Arg (SEQ ID:NO6)Leu-Gly-Leu-Asp-Ser-Glu-Glu-Asp-Tyr-Tyr-Thr-Pro-Gln-Lys-Val-Asp-Val-Pro-Lys(SEQ ID:NO 7)

TABLE 1 Isolation and Purification of ITAK From IL-1α-Induced RabbitLung total protein Spec. Act. protein volume conc. activity (Units/Preparation (mg) (ml) (mg/ml) (Units) mg) fold purif. total lunghomogenate 57300   5700 10.05 [0] 0.43 1 S15Q load (0-25%  1510   20400.74 [0] 16.5 38 ammSO4 pellet) Green 19 load  333   241 1.38 19039 57.1133 Superdex 200 load  112   52 2.15 {5474} 222.1 517 Heparin Sepharoseload   9.4 20 0.47 24851 2646 6153 Mono Q, pH 8.0 load   *1   70 0.0145345 5345 12430 SEC-400 load    0.225 1.35 0.167 4105 18244 42428microMono Q load    0.14 10.6 0.013 6477 46254 107591 microMono Q   $0.10.062 1.61 9844 98640 229395 fraction #12 *estimated, too dilute to getaccurate measurement $estimate based on A @280 nm and silver staining [] unable to assay due to high background and inhibition { } aberrantlylow, SA based on HS load

Example 3 Identification of Human Kinase Gene Sequence in A HumanDendritic Cell cDNA Library

Human dendritic cells (DC) were purified from freshly collected humanbone marrow as follows.

Bone marrow cells were fractionated on a Ficoll density gradient, andCD34+ bone marrow cells were isolated from the high density (buffy coat)fraction using a Ceprate LC CD34 biotin kit (CellPro, Bothell, Wash.)according to the manufacturer's instructions. Briefly, buffy coat cellswere incubated with biotinylated monoclonal anti-CD34 antibody, washedin buffer containing normal saline, and the cell suspension applied to acolumn of solid-phase immobilized streptavidin. CD34+ cells wereadsorbed to the column by streptavidin-biotin affinity binding whileCD34− cells washed through in the column effluent. Positively slectedCD34+ cells were then mechanically desorbed from the flexible column bysqueezing it, because the streptavidin-biotin interaction is of higheraffinity than that between the antibody and its cognate ligand, CD34.

CD34+ cells were cultured at 37° C. in a humidified incubator (10% CO₂)for two weeks in Super McCoy's medium supplemented with 10% fetal calfserum, 20 ng/ml granulocyte-macrophage colony stimulating factor, 20ng/ml IL-4, 20 ng/ml TNF-α, and 100 ng/ml FLT3 ligand. Viable cellsrecovered from cultures were further selected for expression of theknown DC cell surface markers CD1a and HLA-DR by fluorescence-activatedcell sorting (FACS) using antibodies specific for these markers.

Total CD1a+/HLA-DR+ DC RNA was isolated by guanidiniumthiocyanate-cesium chloride gradient centrifugation (standard protocol,see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,Cold Spring Harbor Laboratory Press (1989)). Polyadenylated RNA waspurified on oligo dT-coupled latex beads (Qiagen, Chatsworth, Calif.),according to manufacturer's instructions. A DC cDNA library in plasmidpBluescriptSK(−) (Stratagene, La Jolla, Calif.) was prepared essentiallyas described in Larsen et al., J. Exp. Med. 172:159 (1990). Briefly,approximately 1 μg of polyA+ DC RNA was converted to double strandedcDNA using random hexamer primers and reverse transcriptase using aTimesaver cDNA kit (Pharmacia, Piscataway, N.J.). The cDNA reactionswere optimized to generate an average cDNA size of about 400 bp. Thedouble-stranded cDNA was modified with BglII adapters (described inLarsen et al., supra) and ligated to pBluescriptSK(−) that had beenlinearized with BamHI and similarly modified with BglII adapters. Therecombinant constructs were transformed into E. coli.

Subtractive hybridization of the human DC cDNA library was conductedusing a human fibroblast cDNA library to enrich for cDNAs preferentiallycontained in the DC library. In brief, the DC cDNA library inpBluescript was converted to single-stranded phagemid, and thesingle-stranded phagemid was subtracted with biotinylated RNAtranscribed from the inserts of a driver cDNA library prepared fromhuman foreskin fibroblasts in a modified λgt10 vector containing an SP6RNA polymerase promoter. For a general description of the methodsinvolved, see Klar et al. (Cell 69:95 (1992)) and Owens et al. (Mol.Cell. Biol. 11:4177 (1991)). Single-stranded phagemids recovered fromsubtraction were retransformed into E. coli. Individual colonies wereisolated, and plasmid DNA was prepared and sequenced using dyeterminator methodology (ABI Prism DyeDeoxy Kit, Perkin-Elmer, FosterCity, Calif.).

Each plasmid was sequenced in one direction using a vector-specificprimer adjacent to the BamHI cloning site. Sequences were compared tonon-redundant protein and nucleotide database sequences (National Ctr.for Biotechnol. Information (NCBI), Bethesda, Md.) using the BLASTalgorithm. (Altschul et al., J. Mol. Biol. 215:403, 1990. ) Translationof the ITAK cDNA insert of clone HH0381 (542 nt) revealed that itencoded the GAFGEATLYR amino acid sequence (SEQ ID:NO 4) previouslydetected in a rabbit lung ITAK tryptic peptide. (See Example 2.) Thetranslated HH0381 sequence also showed homology to catalytic domains ofseveral protein kinases in the NCBI database. The HH0381-encoded partialITAK amino acid sequence showed the strongest sequence homology (greaterthan 30% identity) with a corresponding region of the murine nek1protein (Letwin et al., EMBO J. 11:354, 1992), among protein kinasesequences in the database.

Example 4 Cloning of Full Length Gene Encoding ITAK

To identify the full length human gene encoding ITAK, the HH0381 humancDNA cloned insert was ³²P labeled by random priming according tostandard procedures (Sambrook et al., supra) for use as a probe toscreen a human dendritic cell cDNA library prepared using the λZAPIIvector (Stratagene, La Jolla, Calif.) according to the manufacturer'srecommendations. Briefly, the cDNA insert of clone HH0381 (542 bp) wasexcised from the plasmid, gel-purified, radiolabeled with ³²P using aPrime-It II kit (Stratagene, La Jolla, Calif.), and used to probeanother DC cDNA library prepared in bacteriophage lambda vector λZAPII(Stratagene). This second DC cDNA library was prepared from the same DCmRNA as the DC library described above, except a cDNA fraction having alarger average size (about 1000 bp, instead of 400 bp) was used. ThecDNA ends were modified with EcoRI adapters included in the TimesavercDNA synthesis kit (Pharmacia), and the ligated to EcoRI-digestedλZAPII. One positive clone was selected on the basis of hybridization tothe HH0381-derived probe and isolated by successive rounds ofpurification and re-hybridization. The insert of this clone, designatedclone 7, was sequenced using dye terminator methodology.

FIG. 1 (SEQ ID:NO 8) shows a composite nucleotide sequence of theITAK-coding strands of the cDNA insert of clone 7 (nucleotides 1-2040),and clone 16-1 (nucleotides 2041-3264). The translated amino acidsequence (640 amino acids) of the open reading frame is depicted belowthe corresponding nucleotide sequence. Examination of the clone 7 insertDNA coding sequence showed that upon translation into an amino acidsequence, it encoded the kinase signature peptide sequence GRGAFGEATLYR(Hanks et al., Science 241:42, 1988), a portion of which, GAFGEATLYR,had been identified in a rabbit ITAK tryptic peptide as SEQ ID:NO 4.(See Example 2.) The amino acid sequences of additional rabbit ITAKtryptic peptides were also found to be encoded by portions of the humanclone 7 DNA sequence, including the ITAK peptides of SEQ ID:NOS 4-7.While clone 7 included sequences encoding an initiator methionineresidue, it did not appear that clone 7 contained DNA sequences encodingthe full ITAK open reading frame (ORF).

In order to identify the sequences encoding the remainder of the ITAKORF, a new DNA probe was designed from the clone 7 sequence data for usein hybridization to additional human cDNA libraries. A 918 bp DNA probewas prepared from the clone 7 insert sequence as follows: First, thefragment was amplified by polymerase chain reaction (PCR) using theindicated primers:

a) 5′ primer: CCATGGCTGAGACGCTTG (SEQ ID:NO 9) b) 3′ primer:GTCGTCCATATTCGCCACAG (SEQ ID:NO 10)

Template DNA (˜2×10⁶ phage) consisted of a human cDNA library made inλgt10 from the human epidermal carcinoma cell line KB (ATCC CCL17). A 50μl amplification reaction contained template (˜2×10⁶ phage) plus 25 pmolof each primer, 10 mM Tris-HCl, pH 8.3, 1.5mM MgCl₂, 50 mM KCI, 200 μMeach dATP, dGTP, dCTP, dTTP, and 2.5 units of Taq polymerase (BoehringerMannheim, Indianapolis, Ind.). Reactions conditions were: 1 cycle of (5min, 94° C.; 1 min, 64° C.; 2 min, 72° C.); 29 cycles of (1 min, 94° C.;1 min, 64° C.; 2 min, 72° C.); followed by 5 min, at 72° C.

Second, a probe was made from this amplified, clone 7-derived fragmentby using 7.5 ng of the fragment as template in a 100 μl amplificationreaction containing 50 pmol of the 3′ primer (GTCGTCCATATTCGCCACAG)(primer (b) above; (SEQ ID:NO 10)), 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂,50 mM KCl, 20 μM each dATP, dGTP, dTTP, 1 μM dCTP, 100 μCi [α-³²P]dCTP,and 5 units of Taq polymerase (Boehringer Mannheim). Reactionsconditions were: 5 min, 94° C.; followed by 29 cycles of (1 min, 94° C.;1 min, 55° C.; 1 min, 72° C.); followed by 5 minutes at 72° C.Unincorporated radioactivity was removed by passing the probe overSephadex G-50 (Pharmacia).

The radiolabeled 918 bp probe was then used to screen 500,000 plaquesfrom a human dermal fibroblast library made in λgt10 (Sims et al., Proc.Nat. Acad. Sci. USA 86:8946, 1989). Multiple (≧60) positive plaques wereidentified. Approximately 20 of the positive primary plaques were pickedand analyzed by amplification with a combination of primers derived fromthe clone 7 sequence and primers from the λgt10 vector. The primers usedwere:

c) CAACCAGTGAGTCATCCTC (directed toward the 5′ end of the mRNA) (SEQID:NO 11) d) CAACCATGAAGCATACCATG (directed toward the 3′ end of themRNA) (SEQ ID:NO 12) e) CGAGCTGCTCTATAGACTGCTGGGTAGTCC (vector primer,left arm) (SEQ ID:NO 13) f) TAACAGAGGTGGCTTATGAGTATTTCTTCC (vectorprimer, right arm) (SEQ ID:NO 14)

Analysis of the sizes of the amplification products generated usingprimers (d), (e) and (f) revealed that clones designated 11-1 and 16-1could be expected to contain the remainder of the ITAK coding region (onthe C-terminal side), which was not present in clone 7. This conclusionwas verified by direct DNA sequencing of the PCR products from these twoclones.

DNA sequence analysis of clones from fibroblast cells and from dendriticcells revealed several variant sequences. For example, nucleotide 419 inFIG. 1A (SEQ ID NO: 8) is an “A” in clones derived from dendritic cells(clones 7 and 2) and a “T” in clones derived from fibroblast cells. Thisnucleotide change is silent, however, the codon results in an Ile inboth cases. In addition, for nucleotide 443, a “C” is found in dendriticcell clones 7 and 2; a “T” is found in fibroblast clones 3 and 16. Thisvariant is silent also. A non-silent variant is found at nucleotide1405; fibroblast cell clones 3, 11, and 16 have an “A” (His codon)whereas dendritic cell clones 2 and 7 have a “G” (Arg codon).Furthermore, clone 16 has a 36 base insertion at nucleotide 1649. Thissmall insertion appears to be an intron that is normally spliced out ofthe mature mRNA. The source of the other described variant positions islikely to be natural polymorphisms. It is unlikely that thesealterations were introduced during cloning as each variant was found inat least two independently derived clones.

Thus, a composite of clones 7 and 11-1 encodes an entire open readingframe of ITAK. FIG. 1. The open reading frame is 979 amino acids inlength. The ITAK domain with homology to protein serine/threoninekinases lies in the N-terminal ≈300 amino acids (amino acids ≈50-300).The closest relative is a kinase called nek1 (GenBank accession numberS25284). The ITAK domain corresponding to amino acids ≈300-750 hashomology to a family of guanine nucleotide exchange factors for the lowmolecular weight G proteins ran and TC4. The closest relative is calledRCC1 (GenBank accession number A26691, Bischoff and Ponstingl, Nature354:80, 1991).

DNA sequencing was performed using dye-terminator chemistry and customprimers on ABI/Perkin Elmer 373 and 377 automatic DNA sequencers.

Example 5 Direct Polymerase Chain Reaction Cloning of ITAK from cDNALibraries

A cDNA containing the entire coding region for the ITAK polypeptide isamplified from cellular RNA in a form suitable for subcloning into anappropriate vector. RNA from an appropriate cellular source known toexpress ITAK, for example human dendritic cells, human dermalfibroblasts or KB cells (see Examples 3 and 4), is used as template forfirst strand cDNA synthesis. Briefly, 1-5 μg of total RNA is mixed with0.5 μg of oligodT₁₂₋₁₈ primer in 12 μl final volume and heated to 70° C.for 1 min, then chilled on ice. To the above mixture are added 2 μl 1033PCR buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl), 2 μl 25 mM MgCl₂, 2 μl10 mM mixed dNTPs (10 mM each dATP, dGTP, dCTP, and dTTP), and 2 μl 0.1M dithiothreitol, and the reaction is allowed to proceed for 5 min at42° C. Superscript RTII reverse transcriptase (200 units) (GibcoBRL,Gaithersburg, Md.) is added to the reaction, which proceeds for 50 minat 42° C. and is halted by incubation at 70° C. for 15 min, after whichthe mixture is held on ice. RNase H (4 units) (GibcoBRL) is added to thereaction and incubated for 20 min at 37° C.

To generate ITAK-encoding cDNA, 2 μl of the first strand cDNA is used asa template in a 50 μl polymerase chain reaction (PCR) containing 25 pmolof each primer, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 200 μMeach of dATP, dGTP, dCTP, and dTTP, and 2.5 units of Taq polymerase(Boehringer Mannheim, Indianapolis, Ind.). Reaction conditions are: 1cycle of (5 min 94° C.; 1 min 64° C.; 2 min 72° C.); 29 cycles of (1 min94° C.; 1 min 64° C.; 2 min 72° C.); followed by 5 min at 72° C.Suitable primers would contain the following sequences:

a) 5′  primer: ATGTCGGTGCTGGGCGAG (SEQ ID:NO 15) b) 3′  primer:CTAGAGGCTGGGTCTACAG (SEQ ID:NO 16)

In order to clone the ITAK coding segment in a vector suitable formammalian expression, for example the expression plasmid pDC304 (sfNCAV,Immunex, Seattle, Wash.) or other expression plasmids well known in theart, the isolated PCR product fragment is ligated into a vector that hasbeen cut with a suitable restriction enzyme, for example Not1 in thecase of pDC304, and that has had the restriction site subsequentlyblunt-ended by filling in with T4 DNA polymerase and dNTPs (Sambrook etal., supra). Alternatively, the primers may be synthesized with, forexample, Not1 restriction sites on their 5′ ends:

a) alt. 5′  primer: ATATGCGGCCGCATGTCGGTGCTGGGCGAG (SEQ ID:NO 17) b)alt. 3′  primer: ATATGCGGCCGCCTAGAGGCTGGGTCTACAG (SEQ ID:NO 18)In this instance, the isolated PCR product is digested with Not1 andligated into the Not1-cut vector without the intermediate end-fillingstep.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of screening for an agent that modulates the kinase activityof an IL-1/TNF-α-activated kinase (ITAK), comprising: (a) contacting acandidate agent with biologically active ITAK under conditions and for atime sufficient to allow the candidate agent to modulate the kinaseactivity of said ITAK; and (b) measuring the ability of the candidateagent to modulate the ITAK kinase activity, wherein saidIL-1/TNF-α-activated kinase is a protein comprising an amino acidsequence that is at least 80% identical to SEQ ID NO:2.
 2. The method ofclaim 1, further comprising isolating the candidate agent.